JP2525436C - - Google Patents

Description

Description: FIELD OF THE INVENTION The present invention relates to the production of proteins by cell culture, wherein the cells producing the protein are cultured in the presence of a chemical agent that increases the production of the protein. BACKGROUND OF THE INVENTION Culturing cells for the purpose of industrially producing cell products for diagnosis and therapy is an expensive and time consuming method. The equipment required is expensive and the research, development and manufacturing costs are high. For an industrially viable process, it is desirable to use a cell line that produces large quantities of the product. Many naturally occurring cells do not produce large quantities of the desired protein, but naturally produce small quantities of the desired protein and are constantly synthesizing proteins. Although other cell lines do not inherently produce the protein of interest, it is well known that such cell lines can be induced to produce the protein of interest. That is, these cell lines have the potential to produce specific proteins and produce these proteins when given an appropriate stimulus (Kruh, J .: Mol. Cell. Biol., 42: 65-65). 82, 1982
). Induction of protein production can be achieved by adding to the culture medium a chemical compound such as dimethyl sulfoxide (DMSO) or an infectious agent such as Sendai virus to the cells. Many compounds are known that can act as "inducers,""enhancingagents," or "stimulants" for protein production in cell lines. In this book, the above terms are:
It shall have the following meaning based on the apparent effect it manifests. “Inducer”
Refers to an agent that clearly induces or initiates protein production. "Enhancer"
An agent that clearly shows an effect of increasing protein production but does not induce or initiate protein production. The enhancer may be added to the cell culture during or immediately after the induction of the cell line, or may be added at any time to the cell line that is continuously producing the desired protein. A "stimulant" is added to a culture of an inducible cell line, usually at a time when the cells have reached a desired cell density but have not yet induced the production of protein, and are apparently An agent that acts as part of the triggering mechanism for inducing protein production. Whether an inducer, enhancer, or stimulant, the choice of reagent depends on the type of cell,
It depends on various factors such as the final concentration of the reagent added to the medium, the time of addition, the duration of exposure to the reagent and the toxicity. To date, many additives have been used, such as DMSO, urea derivatives and alkanoic acids or salts. Among such additives, sodium butyrate has recently been particularly studied. This compound has been shown to produce various morphological and biological modifications in a reversible manner upon addition of millimolar concentrations to cultures of various natural or selected cell lines (Kruh, J. et al. :
Supra). At the molecular level, butyrate is believed to cause histone hyperacetylation by inhibiting histone deacylase. In general, butyrate modulates gene expression, and in almost all cases, addition to cells in culture appears to arrest cell growth. For example, sodium butyrate (1 mM) has been used to induce the synthesis of human chorionic gonadotropin in HeLa cell cultures (Ghosh et al .: Biochem.
J., 166: 265-274, 1977; Cox & McClure: In Viro, 19: ^# 1.
. 1-6, January 1983), but in each case butyrate treatment inhibits cell growth. UK Patent Specification No. 2122207A also discloses that Se in lymphoblastoid cell lines
The use of butyrate as a stimulant has been described prior to the induction of interferon synthesis in the ndai virus. However, British Patent No. 2122207A
The article further discloses that, for the particular cell lines described, butyrate cannot be used as an enhancer of interferon synthesis when added at or shortly after induction. In addition to cell lines that need to induce large-scale production of the protein of interest, there are other cell lines that can produce the protein originally desired without the use of inducing agents (Trends Bi
otech., 3: No. 12, 1985; Immuno. Meth., 56: 221-234, 1983.
). Such cell lines include genetically engineered cell lines and hybridoma cell lines. The use of selected genetically engineered and hybridoma cell lines allows for the production of large amounts of rare proteins without the use of inducing agents. The effects of butyrate, which has been recognized as an inducer of protein production in several mammalian cell lines, have been studied for the culture of certain genetically engineered cell lines. For example, the entire SV40 genome and the herpes simplex virus thymidine kinase (HSV-T
Culture medium of Syrian hamster cells obtained by microinjection or transfection of a plasmid containing the gene encoding K) is treated with butyrate (Yuan et al .: J. Biol. Chem., 260: 3778-3783). , 1985
). Butyrate was added to the cell culture to maintain butyrate concentration throughout the culture. S
The expression level of V40 was determined by quantification of the amount of SV40T antigen produced.
The level of SV-TK expression was measured by tritiated thymidine incorporation. Yuan et al. Report that butyrate at concentrations ranging from 1 mM to 5 mM inhibited expression of both genes as compared to control values. Furthermore, other researchers (Gorman, CM et al .: Nucl. Acids Res., 11: 7631)
7648, 1983) study the effect of sodium butyrate on DNA-mediated gene transfer in examining the relationship between chromatin structure and expression of recombinant plasmids. In both transient and stable transformation experiments, cells were treated with butyrate immediately after transfection with a plasmid containing exogenous DNA. In the case of stable transformants, a second butyrate treatment was performed 5 weeks after the first butyrate treatment immediately after the transfusion. These results indicate that butyrate affects the early stages of gene activity at at least two levels, increasing both the percentage of cells capable of expressing exogenous DNA and the level of enhancer-dependent transcription. I have. Furthermore, the results of experiments with stable transformants show that the expression of the inserted recombinant plasmid gene was re-induced by subsequent butyrate treatment when cells were initially treated with butyrate immediately after DNA uptake. It is shown that it is done. In the cells that had not been subjected to the initial butyrate treatment immediately after transfection, no induction of the expression of the exogenous gene was observed. These results suggest that the initial butyrate treatment creates a basis for inserted foreign DNA expression for subsequent butyrate induction. In summary, butyrate has previously been added to the cultures of many natural or engineered cell lines, but in general, the addition of butyrate initiates or inhibits gene expression,
It appears to stop cell growth. In engineered cells, prolonged butyrate treatment appears to inhibit the expression of foreign genes (Yuan et al., Supra). In the special case where cells were treated with butyrate immediately after transfection, subsequent butyrate treatment appears to re-induce expression of the foreign gene (Gorman et al., Supra). No studies have been made on growth and immunoglobulin production in hybridoma cell culture. The present invention provides the ability to increase the production of a protein during genetic engineering and culturing of hybridoma cells, which are cells that naturally produce a certain protein, by adding an appropriate concentration of a chemical agent such as butyrate to the culture medium. Discovered and completed. In the case of genetically engineered cells, initial treatment with the reagent immediately after transfection is not necessary to increase protein production levels by treating the cells with a reagent such as butyrate during culture. Was. In particular, it has been unexpectedly discovered that it is possible to add an agent such as butyrate to the culture of hybridoma cells at a concentration that increases protein production but does not substantially reduce cell growth. Was. In addition, high levels of reagents such as butyrate inhibit the growth of genetically engineered cells and hybridoma cells, but use reagents at concentrations that increase protein production and do not substantially reduce cell viability. It also revealed what they could do. Accordingly , the present invention provides, as a first aspect, a method for producing a protein,
Culturing genetically engineered cells or hybridoma cells that naturally produce the protein in the presence of an agent that increases the production of the protein but at a concentration that increases the production of the protein but does not substantially reduce the growth rate of the cells. A method of characterizing is provided. In yet another embodiment of the present invention, (a) an agent that increases the production of a protein is present at a concentration that increases the production of the protein but does not substantially reduce the growth rate of the cells, thereby naturally producing the protein. The present invention provides a method for obtaining a protein comprising the steps of (b) continuing culturing until the protein accumulates, and (c) isolating the protein if desired. In the present invention, a cell that naturally produces a protein should be understood as a cell that does not need to induce the production of the protein. The term "engineered cell" refers to a cell that has been transfected or transformed with exogenous DNA encoding the desired protein to provide a transformed cell that naturally produces the desired protein. Such genetically engineered cells include cells whose exogenous DNA is maintained as extrachromosomal genes inside transformed cells or cells whose genome is inserted into the genome. Suitable host cells for the preparation of such engineered cells include all types of host cells, but are usually yeast cells or, preferably, eukaryotic cells, including animal cells such as mammalian cells. The term "hybridoma cell" generally refers to a hybrid cell produced by fusing an antibody-producing cell, such as a B lymphocyte, with a suitable myeloma cell. However, as used herein, the term "hybridoma cells" also includes "immortalized" antibody-producing cells obtained by treating antibody-producing cells, such as B lymphocytes, with a transforming agent, such as Epstein-Barr virus (EBV). I do. The "hybridoma cell" in the present invention is usually of animal origin, for example, a mouse or rat hybridoma cell or a human cell-derived hybridoma cell. The protein produced by the method of the present invention may be a recombinant protein. For example, useful and / or therapeutic eukaryotic proteins, ie, growth hormones such as hormones, eg, human growth hormone, enzymes, eg, tissue plasminogen activators, enzyme inhibitors, eg, tissue inhibitors of metalloproteinases or lymphokines, etc. The protein may also be an immunoglobulin molecule, especially when the cell is a hybridoma cell, including a naturally occurring antibody molecule or analog thereof, or a portion of any of these. For example, natural or The method of the present invention is particularly useful for the production of immunoglobulins from hybridoma cells.The reagents used to increase protein production in the method of the present invention (hereinafter referred to as "FAB fragments"). , Enhancers) are chemical agents that can be used at a concentration that generally increases protein production but does not substantially reduce cell growth rate. It is preferably selected from the group consisting of unsaturated fatty acids, especially alkanoic acids and their salts, in particular the enhancer is a linear, straight-chain 1 to 10 carbon atoms in length, particularly preferably 3 to 6 carbon atoms. Alkanoic acid or a salt thereof, although the above statement is not excluded, compounds having an even number of carbon atoms are stronger than compounds having an odd number of carbon atoms. Of seems that. Enhancers The acid or its salts especially alkali metal salts, are especially sodium butyrate for example butyrate are preferred. Enhancers, 0.01MM～500mM in culture Mejiumu, preferably 0.1mM~2
It is present at a concentration of 00 mM, particularly preferably 0.2 mM to 10 mM. For transformed cell lines and other mammalian cell lines, the most preferred concentration range is 1 mM to 10 mM. In the case of a hybridoma cell line, the concentration range is preferably 0.1 mM to 0.9 mM, more preferably 0.3 mM to 0.7 mM, and particularly preferably 0.4 mM to 0.6 mM. However, it is envisaged that the concentration of enhancer used will vary, taking into account the particular cell line to be cultured. The most appropriate concentration of enhancer for a particular cell line must be determined in accordance with routine practice, and by means of suitable and small-scale tests. According to a preferred embodiment of the present invention, the present invention provides a method for obtaining a protein by culturing a hybridoma cell, wherein (a) an agent for increasing the production of a protein is used to increase the production of the protein, but the growth rate of the cell is reduced. In the presence of a concentration that does not substantially reduce, the hybridoma cells that naturally produce the protein are cultured, (b) culturing is continued until the protein is accumulated, and (c) from each step of isolating the protein, if desired. Provide a way to become. In this preferred embodiment, the hybridoma cells are preferably mouse or rat hybridoma cells. The protein produced by this method is preferably an immunoglobulin. The enhancer is preferably an alkanoic acid or a salt thereof, preferably a linear C ₂
ＣC ₁₀ , especially C ₃ -C ₆ alkanoic acids or salts thereof, especially butyric acid or salts thereof, especially alkali metal salts such as sodium butyrate. No enhancer was added in the culture medium.
. 1 mM to 0.9 mM, more preferably 0.3 mM to 0.7 mM, particularly preferably 0 mM
. It is present in a concentration range from 4 mM to 0.6 mM. For culturing cells in the method of the present invention, any suitable culturing operation and culturing medium can be used. Suitable culture procedures are well known and understood by those skilled in the cell culture art (eg, J. Immun. Meth., 56:22).
1-234, 1983). Both serum-supplemented and serum-free media can be used. Both fermentation operation by batch method and continuous method, suspension culture and adherent culture such as microcarrier culture method, stirring tank and pneumatic fermentation tank, etc.
An appropriate operation can be adopted depending on the type of the cell. In the method of the present invention, the enhancer is added to the culture medium before or after adding the cells to the culture medium. If desired, the enhancer can be added more than once. For example, it may be desirable to add the enhancer early in the culture, and then add more enhancer as the culture proceeds. Of course, the addition of the enhancer must be performed with sufficient control so that the concentration does not exceed the concentration that may decrease the growth rate of the cells. The production of the protein in culture can be monitored by conventional quantification techniques, for example by an enzyme-linked immunosorbent assay or an immunoradiometric assay appropriate for the particular protein in question. The desired protein can be isolated from the cell culture as needed using conventional separation techniques. For example, proteins in culture media are separated from cells by centrifugation,
The supernatant containing the protein is collected and then concentrated, for example by ultrafiltration. The protein thus obtained can be further purified, if desired, using a conventional protein purification method. If the desired protein is not secreted into the culture medium during the culture, the cells are destroyed, and the protein is obtained by treating the cells as described above. The enhancers described above can also be used to enhance protein production in cells that have arrested logarithmic growth and are in stationary or declining growth. This is particularly advantageous if the desired protein is produced by the cell only during these late two phases. According to another aspect of the present invention, the present invention provides a method for producing a protein, wherein the agent that increases protein production increases the protein production but does not significantly reduce cell viability (eg, (Substantially non-toxic to such proteins) at a concentration to maintain genetically engineered cells or hybridoma cells that naturally produce the protein in culture. The cells, enhancers and culturing procedures used in this aspect of the invention can be as detailed above. The culture medium used preferably supports the viability of the cells, but does not activate the growth of the cells. Such a medium is a so-called maintenance medium and does not contain, for example, essential growth factors such as transferrin, insulin, albumin and the like. In this aspect of the invention, cells are usually grown in culture until they reach the level of biomass required for efficient protein production. In this case, if desired, an enhancer can be included in the growth medium; however, in some cases, for example, where protein production has a deleterious effect on the cell, the cell is grown without the enhancer. It is desirable. That is, cells are grown to or near the maximum cell density in the case of suspension culture, or to or near confluency in the case of adherent cell lines. Once the cells have been transferred to the maintenance medium, the enhancer is added at a concentration that increases protein production but does not significantly reduce cell viability. In general, suitable concentrations of the enhancer are as described above, for example from 0.1 mM to 500 mM, more preferably from 0.1 mM to 200 mM, particularly preferably from 1 mM to 5 mM. The exact concentration depends on the cells used. In yet another embodiment, the present invention provides a method for producing a protein, comprising the steps of, in a first stage, transforming a genetically engineered cell or hybrid cell that naturally produces the protein into a growth medium to a predetermined cell density. Let it grow and then the second
At this stage, there is provided a method of maintaining a cell in a medium in which an agent that increases protein production is present at a concentration that enhances protein production but inhibits cell growth without reducing cell viability. This embodiment of the invention, particularly when applied to genetically engineered cell lines that are moderately or microproducer cells of the desired protein, can significantly increase protein production by use of appropriate concentrations of enhancers. . An agent for increasing the production of protein may be added to the growth medium, but is preferably not added. The predetermined cell concentration for growing cells in the first stage is preferably a cell concentration close to the maximum cell density in culture or close to confluence.
The concentration of the enhancer used in the second step is generally in the range described above, but in particular, the concentration that stops cell growth, prolongs cell viability retention time, and increases total protein production level Can also be used. As outlined above, the advantage of adding a chemical agent, such as butyrate, to the culture system is that the production of protein can be substantially increased with very little expense, and even with changes in culture technology. It's very slight. Yet another advantage is that the production protocol is simple, since the cells are naturally cells that produce the desired protein and do not require induction of mass production of the desired protein, and that the appropriate concentration depends on the cell. The addition of a range of butyrate will increase protein production.

BRIEF DESCRIPTION OF THE DRAWINGS The invention will be further described by the following examples with reference to the drawings, which are merely illustrative of the invention and do not limit the invention in any way. . FIG. 1 is a graph showing the effect of butyrate on the growth of hybridoma cells in medium without serum. FIG. 2 is a graph showing the effect of butyrate on hybridoma cell growth and antibody production in an air-fed fermenter. DESCRIPTION OF SPECIFIC EMBODIMENTS Example 1 Effect of Sodium Butyrate on Hybridoma Cell Growth and Antibody Production IgM-secreting mouse hybridoma cell line NB1 / 19 was added to albumin, insulin, transferrin, gentamicin and other components necessary for suspension culture of cells. Were grown in suspension in Dulbecco's Modified Eagle Minimal (DMEM) serum-free medium containing the same. The NB1 / 19 cell line produces a mouse monoclonal antibody that has specificity for the antigenic determinants of human B blood cells. The preparation of this cell line is described in detail in GB 2097425B. However, it should be understood that other hybridoma cell lines could be used as appropriate in this and other examples, as an alternative to this particular cell line. Cells were grown to a viable cell density of 1.0-1.5 × 10 ⁶ cells / ml. Viable cell density was determined by counting the number of cells that excluded the stain trypan blue. A sample of the suspension culture was added to a serum-free medium at about 1.3 × 10 ⁵ cells / m 2.
Dilute to a density of l. Aliquots of 50 ml were dispensed into shake flasks and sodium butyrate was added to concentrations of 0.1 mM, 0.3 mM, 0.5 mM and 1.0 mM. Shake flasks are aerated with 5% carbon dioxide-95% air, stoppered, and
Incubation was carried out with shaking up to 07 hours (Table 1). A sample (1.0 ml) was removed from the culture with a pipette. 100 μl was used to determine the number of viable cells, and the rest was centrifuged at 300 × g for 5 minutes. The supernatant was stored at −20 ° C. until quantification of the antibody by enzyme-linked immunosorbent assay (ELISA). FIG. 1 and Table 1 show the effects of various concentrations of butyrate on hybridoma cell growth and antibody production, respectively. Example 2 Butyrate Antibodies in Hybridoma Cells Cultured in Serum-Fed Medium
Increased Production The protocol for this experiment is the same as in Example 1 except that the cells were cultured in Dulbecco's modified Eagle's minimal medium supplemented with 10% fetal calf serum. Table 2 shows the effect of sodium butyrate addition on antibody production. Example 3 Butyrate Production of Antibodies in Hybridoma Cells Cultured in Pneumatic Fermenters
Increase NB2 / 19 hybridoma cells pneumatically fermentation tank (Birch addition: Trends Biotech
. 3: No. 7,162 to 166) (5l), glutamine, transferrin,
Suspension culture was performed using serum-free DMEM medium containing albumin, insulin and the like. Hybridoma cells were inoculated into the fermentor at a cell density of 1 × 10 ⁵ cells / ml. When the fermenter was inoculated with hybridoma cells, sodium butyrate was added to the fermenter to a concentration of 0.5 mM. Controls did not receive sodium butyrate. Samples were taken periodically as described in Example 1. The effect of butyrate on cell growth and antibody production is shown in FIG. Example 4 Increased Production Using Recombinant Human Growth Hormone Sodium Butyrate in Mammalian Cells Molecular biology of adherent CBMG human growth hormone producing cells is described in C.W. N. Pa
vlakis et al., PNAS (USA), 80: 397-401, 1983. CBMG cells were seeded at 0.7 × 10 ⁵ cells / cm ² in Dulbecco's Modified Eagle's Minimal Medium (DMEM) supplemented with 10% fetal calf serum in tissue culture flasks. After the cells in the culture had grown to confluence, the DMEM serum supplemented medium was replaced with a serum-free DMEM maintenance medium. After transferring the cells from the serum-supplemented medium to the maintenance medium,
Sodium butyrate was added at 1 mM and 5 mM. Human growth hormone levels in the culture supernatants were measured after 48 hours by immunoradiometric assay. The results are shown in Table 3. Example 5 Effect of sodium butyrate on various hybridoma cell lines Serum-free medium (see Example 1) or heat-inactivated fetal calf serum 10% (v
Hybridoma cells were grown in suspension culture in any of DMEM supplemented with (v). Serum was replenished to serum-free media as shown in Table 4. Cells were cultured in spinning bottles or shake flasks as in Example 1. Sodium butyrate was added to the culture immediately after inoculation. Butyrate treatment increased antibody production per cell. Table 4 shows that for a number of different hybridoma lines, butyrate treatment increases harvest antibody titers. Example 6 Effect of various alkanoates on antibody production from hybridoma cells NB1 / 19 cells were cultured as in Example 1. Various linear sodium alkanoates (propionate, butyrate, pentanate or hexanoate)
Was added to the inoculum culture at the concentrations shown in Table 5. Table 5 shows that alkanoate treatment increases the production capacity of hybridoma cells. Patterson edition. (See Academic Press, 1973) by counting nuclei released from cells. The concentration of tPA in the supernatant was quantified using the Fibrin-Agar method. Example 7 Increased Production of Recombinant tPA from Cell Line by Butyrate Treatment Recombinant Tissue Plasminogen Activator (tPA) Producing Mouse C127
The cell lines were placed in a rotating vessel (Bellco) in a microcarrier (Geri beads, KC. Biolo).
gicals). The growth medium was DMEM supplemented with 10% fetal calf serum. The microcarrier concentration was 5 g (dry weight) per liter of media. Cells were seeded at a density of 1 × 10 ⁵ per ml. After the cells grew to confluence on the surface of the microcarrier, the growth media was aspirated and replaced with a protein-free DMEM basal medium containing butyrate. Butyrate was not added to the control culture. The maintenance medium was replaced with fresh maintenance medium (containing butyrate) at the times indicated in Table 6. Cell density is determined by hypotonic degradation (“Tissue Culture Methods and Applications”, Kr.
use &

Claims (1)

[Claims] 1. A method for obtaining tissue plasminogen activator (tPA) by cell culture, comprising the steps of: (a) generating a genetically engineered cell capable of structurally producing the tPA from 2.5 mM to 5 mM
(B) culturing until the tPA accumulates; and (c) optionally isolating the tPA. 2. The method according to claim 1, wherein the alkanoic acid or a salt thereof is butyric acid or a salt thereof. 3. 3. The method according to claim 2, wherein the alkanoic acid or a salt thereof is sodium butyrate.